Tuberculosis (TB) remains an infectious disease with a high globally mortality. Standard chemotherapy for drug susceptible TB requires for at least 6 months. Such lengthy treatment regimens reduce patient adherence leading to treatment failure and drug resistance. The biological mechanisms underpinning the requirement for lengthy TB treatment are complex. A research priority is to understand how and why a small fraction of bacilli, antibiotic survivors , can remain viable in humans for months under treatment. Antibiotic survivors (often referred to as tolerant or persisters) have been studied in vitro, and are a small subpopulation of cells that survive an antibiotic challenge despite genetically encoded susceptibility. They are difficult to study, especially in complex models relevant for TB because they are very rare and heterogeneous. The long-term goal is to understand the genetic mechanisms responsible for antibiotic survival in M. tuberculosis (Mtb) in the most biologically relevant systems using innovative approaches. Particularly as there is evidence that the host environment can modify mechanisms of antibiotic survival. To overcome the challenges of rarity and heterogeneity the applicants laboratory has developed a inducible dual-color fluorescent reporter system (IDCRS) that can be combined with fluorescent activated cell sorting (FACS) to isolate single cells of Mtb that remain transcriptionally and translationally active even after prolonged antibiotic treatment. IDCRS can also be used to isolate Mtb from infected animals. Using this system it was possible to identify a population of antibiotic survivors that are not culturable but clearly metabolically active. Importantly this population was heavily selected for by specific antibiotics. The main hypothesis is that the mechanisms of antibiotic survival are not universal but are determined by the antibiotic class and host environment. This will be explored through two aims. Aim one is to identify antibiotic survival mechanisms of Mtb in vitro and during infection. The IDCRS will be used to purify antibiotic survivors and then a comprehensive mutagenesis strategy will be deployed to compare the underlying molecular determinants of antibiotic survivors in vitro and in vivo. Aim two is to determine the fate and role of poorly culturable antibiotic survivors during infection. This will be done by FACS purification of antibiotic survivors that are both metabolically active and poorly culturable. Their ability to regrow will then be characterized in vitro under multiple environmental conditions. Then their disease causing potential during infection of a murine model of TB disease will be evaluated by using an individually barcoded library of Mtb. The proposed studies will identify new mechanism of antibiotic survival for Mtb and determine if distinct pathways leading to antibiotic survival exst for different drugs and how the host environment modifies antibiotic survival during infection.